Revision as of 10:28, 14 March 2012

In this tutorial, we will learn how to run a molecular dynamics simulation of a protein-ligand complex. We will then post-process that simulation by calculating structural fluctuations (with RMSD) and free energies of binding (MM-GBSA).

I. Introduction

AMBER

Amber - Assisted Model Building with Energy Refinement - is a suite of about multiple programs for perform macromolecular simulations. Amber11, the current version of Amber, includes newly released functionality such as PMEMD, particle mesh Ewald MD and soft-core Thermodynamics Integration MD. For the tutorial, we are using an older version which is AMBER10.

The Amber 10 Manual is the primary resource to get started with Amber10. (Tip: Using Adobe Acrobat to view the file, you can simply search the document for keywords such as the name of a simulation parameter, which saves much time.) In addition, Amber Tools User's Manual serves as another reference while using Amber tools. You can also read the manual for Amber11 on Amber11 and AmberTools Users' Manuals

Here are some programs in Amber

LEaP: an preparing program for constructing new or modified systems in Amber. It consists of the functions of prep, link, edit, and parm for earlier version of Amber.

ANTECHAMBER: in additional to LEap, this main Antechamber suite program is for preparing input files other than standard nucleic acids and proteins.

SANDER: according to the Amber 10 manual, it is 'a basic energy minimizer and molecular dynamics program' that can be used to minimize, equilibrate and sample molecular conformations. And this is the program we mainly use in this tutorial to generate trajectory files of the molecular system.

PMEMD: version of SANDER that has improved parallel scaling property and optimized speed.

PTRAJ: an analysis program for processing trajectory files. One can use ptraj to rotate, translate the structures, evaluate geometrical features and so on.

There is a mailing list you could sign-up for, as an additional resource.

Biotin and Streptavidin

Organizing Directories

While performing MD simulations, it is convenient to adopt a standard directory structure / naming scheme, so that files are easy to find / identify. For this tutorial, we will use something similar to the following:

II. Structural Preparation

Preparation in Chimera

In this AMBER tutorial, we will use the same system with previous DOCK part. Chimera can directly get the structure by its PDB ID 1DF8. To begin with, we need three files under directory 001.CHIMERA.MOL.PREP.

antechamber

A antechamber input file requires all the atom names to be unique and it only uses the first 3 characters as the name. So if we use 1DF8.lig.chimera.mol2 as the input file, it will cause errors("H102" and "H103" will have the same name "H10").

We need to manually rename the atoms. One way is to use the first column numbers to be the atom names. If you are using Vim, the visual block mode can help by selecting a rectangular section of text. We rename the atoms and save the file as 1DF8.lig.mol2 under directory 001.CHIMERA.MOL.PREP.

Please note that you need to be in the tcsh shell for running the which command shown above. Also, if you are using a different version of amber, you might want to change to the correct version by editing the .cshrc file and source it.

Copy parameters of ions to your working directory from the following resource:

Antechamber is a set of tools to generate files for organic molecules, which can then be read into LEaP. The antechamber program itself is the main program of Antechamber package. It can perform many file conversions, and can also assign atomic charges and atom types. In this tutorial, we use antechamber to convert our input mol2 file into files ready for LEaP. In the command line, type:

You can see this file contains information related to the ring system.

Parmchk is another program included in the Antechamber package. After running antechamber, we run parmchk to check the parameters. If there are missing parameters after antechamber is finished, the frcmod template generated by parmchk will help us in generating the needed parameters:

parmchk -i 1DF8.lig.ante.prep -f prepi -o 1DF8.lig.ante.frcmod

Then open the output file 1DF8.lig.ante.frcmod, you will see something like this:

This means all the needed force field parameters are avalaible except there are two missing dihedral parameters, which were assigned a default value.

Next, we need create 3 new input files – “tleap.lig.in”, “tleap.rec.in” and “tleap.com.in”, for the ligand, the receptor, and the complex, respectively. These input files will be used by LEaP to create parameter/topology files and initial coordinate files.

Visualization in VMD

Then, we can load the gas phase ligand file generated by tleap: VMD Main->File->New Molecule
(Please note that in order to visualize in VMD, we need first load the .prm7 file and then load the .rst7 file)

You can compare this with ligand in the water phase by opening 1DF8.lig.water.leap.prm7, 1DF8.lig.water.leap.rst7 at the same time.

2012_AMBER_Tutorial_1DF8_lig_water

In order to see the ligands clearly, we can visualize without hydrogen atoms. VMD Main->Graphics->Representations->

In the Selected Atoms command line, type: all and noh->Click Apply

2012_AMBER_Tutorial_1DF8_lig_water_noH

You can actually see the ligand in water is very different from that in gas phase.

III. Simulation using sander

Minimization and equilibration

Production

Although we run minimization and production in the same script, they

The final stage now we have equilibrated our system is to run a production simulation. We should run the production phase of the simulation using the same conditions as the final phase of equilibration to prevent an abrupt jump in the potential energy due to a change in simulation conditions.

In The above code we see that we are running for 50000 steps (nstlim = 50000), with a time step of dt where dt of 0.001 is 1fs. We are starting the run with no previous velocity information ntx=1, irest=0.
We will print energy output every 500 steps (ntwr=500) and save coordinates every 500 (ntwx=500).
We want the initial temperature to be 298.15K (tempi=298.15) and the reference temperature to be 300K (temp0=298.15).
Initially, we restraint everything except hydrogens with a restraint weight of 1.0, and with later runs we decrease the restraint weight and only the backbone is restrained.

IV. Simulation Analysis

Ptraj

Yuchen

To run the Ptraj analysis, you should first create a separate folder for it under the root directory if you don't have one.

mkdir 004.PTRAJ

Woo Suk

Finally, you want to perform MMGBSA analysis to obtain energetic information about the system. To do this, you need a trajectory file of the gas phase complex (which we already created above and saved as 1DF8.com.trj.stripfit), and want to create two more trajectory files containing just the receptor and just the ligand. To do so, you have to run two more ptraj using the following ptraj input files:

In VMD, we open one of the .prm7 files in 002.ANTE.TLEAP. If you want to visualize the whole complex in gas state, you can open 1DF8.com.gas.leap.prm7 with AMBER7 Parm from 002.ANTE.TLEAP and then 1DF8.com.trj.stripfit with AMBER coordinates from 004.PTRAJ. With these files, you can look at the real-time movement of the complex in the gas state like following snapshot.

protein_gas.png

You can repeat this procedure to observe the real-time movement of the complex in the water state. Only different thing is opening 1DF8.com.wat.leap.prm7 instead of 1DF8.com.gas.leap.prm7. The following snapshot is that of movement in the water phase.

protein_gas.png

In the snapshot above, the water molecules are shown as points.

MMGBSA Energy Calculation

MM/GBSA is the acronym for Molecular Mechanics/Poisson-Boltzmann Surface Area. This part of AMBER combines molecular mechanics (MM) with both the electrostatic (PB) and nonpolar (SA) contribution to solvation . Topology files are needed for the receptor (R), ligand (L), and receptor-ligand complex (RL). The trajectory files (trj) generate the coordinates. Therefore, molecular dynamics is used to generate a set of snapshots taken at fixed intervals from the trajectories. These snapshots are processed to remove solvent and generate the free energy of binding.

Then this script should be sent to the queue, i.e., qsub the script using the commands:

chmod +x run.sander.rescore.csh
qsub run.sander.rescore.csh

V. Frequently Encountered Problems

Barbara

1.If you have a problem opening the structure of your molecule using VMD, be certain that the prm7 file corresponds to the rst7 file. For example, 1df8.lig.wat.leap.prm7 must be matched to 1df8.lig.wat.leap.rst7.

2. The VMD Console window (often hidden behind the visualization screen of your molecule) can be used to determine where VMD has found an error in your typed commands if you are unable to visualize your molecule.

3. If your "rescore" files do not appear when you are running the script: run.sander.rescore.csh, it may be the result of there not being a free node. You job may be sitting on the queue. If you type qstat -u username, you will be able to see if the job is sitting there because a Q will appear.